Proteases play key roles in the regulation of many biological processes in health and disease. Determination of the preferred cleavage sequence for a protease provides insight into its biological functions and aids in the development of specific inhibitors. A general method will be developed for the rapid determination of optimal substrate sequence motifs for proteases by pool sequencing of degenerate cyclic peptide libraries. As an initial project this method will be used to determine the substrate specificities of the caspases, cysteine proteases involved in the regulation of programmed cell death. Specific inhibitors of caspases will be generated by replacing the scissile bond of preferred peptide substrates with uncleavable aminomethlylene and ketomethylene isosteres, as well as the thiol-reactive alpha-ketoamide group. The pooled library sequencing method will also be applied to find optimal substrates which contain D-amino acids and unnatural amino acids, which can be adapted as described to produce inhibitors. Inhibitors will also be generated by screening for natural and unnatural amino acid- containing peptides which bind to caspases but do not dissociate; such studies will employ both larger cyclic peptides, as those used to determine substrate specificites, as well as smaller, more constrained cyclic peptides. Novel inhibitors will be evaluated in vitro against recombinant proteases and in apoptotic model systems based on cell extracts, as well as in cell culture assays for apoptosis.